The present invention concerns immunoassays to antibodies to human T-lymphotropic virus (HTLV-III) [lymphadenopathy associated virus (LAV)], hereinafter referred to as "HTLV III/LAV".
Human retroviruses with tropism for a subset of T lymphocytes positive for the T4 antigen have been isolated with high frequency from individuals with the acquired immunodeficiency syndrome or from persons at high risk of developing AIDS. These viruses have been designated as human T-lymphotropic virus type III (HTLV III) or lymphadenopathy-associated virus (LAV) and are considered to be the causative agent of AIDS (F. Barre-Sinoussi, J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. Vezinet-Brun, C. Rouzinoux, W. Rozenbaum and L. Montagnier, Sience, 220, 868 (1983); L. Montagnier, C. Dauguet, C. Axler, S. Chamaret, J. Gruest, M. T. Nugeyre, F. Rey, F. Barre-Sinoussi and J. C. Chermann, Ann. Virol., 135E., 119 (1984); E. Vilmer, C. Rouzioux, F. Vezinet-Brun, A. Fischer, J. C. Chermann, F. Barre-Sinoussi, C. Gazengel, C. Dauguet, P. Manigne, C. Griscelli and L. Montagnier, Lancet, 1, 753 (1984); R. C. Gallo, S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, G. White, P. Foster and P. D. Markham, Science, 224, 500 (1984); Schupbach, J., M. Popovic, R. V. Gilden, M. A. Gonda, M. G. Sarngadharan and R. C. Gallo, Science, 224, 503 (1984); M. Popovic, M. G. Sarngadharan, E. Read and R. C. Gallo, Science, 224, 497 (1984); D. Klatzmann, F. Barre-Sinoussi, M. T. Nugeyre, C. Dauguet, E. Vilmer, C. Griscelli, F. Brun-Vezinet, C. Rouzioux, J. C. Gluckman, J.-C. Chermann and L. Montagnier, Science, 225, 59 (1984); D. Klatzmann, E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.-C. Gluckman and L. Montagnier, Nature, 312, 767 (1984); A. G. Dalgleish, P. C. L. Beverly, P. R. Clapham, D. H. Crawford, M. F. Greaves and R. A. Weiss, Nature, 312, 763 (1984); J. A. Levy, A. D. Hoffman, S. M. Kraker, J. A. Landis, J. M. Shimabukuro and L. S. Oshiro, Science, 225, 840 (1984)).
Persons infected with HTLV III/LAV usually have in their serum antibodies to one or more viral proteins (Barre-Sinoussi et al., supra; M. G. Sarngadharan, M. Popovic, L. Bruch, J. Schupbach and R. C. Gallo, Science, 224, 506 (1984); Levy et al., supra; V. S. Kalyanaraman, C. D. Cabradilla, J. P. Getchell, R. Narayanan, E. H. Braff, J.-C. Chermann, F. Barre-Sinoussi, L. Montagnier, T. J. Spira, J. Kaplan, D. Fishbein, H. W. Jaffe, J. W. Curran and D. P. Francis, Science, 225, 321 (1984); F. Brun-Vezinet, F. Barre-Sinoussi, A. G. Saimot, D. Christol, L. Montagnier, C. Rouzioux, D. Klatzmann, W. Rozenbaum, J. C. Gluckmann and J. C. Chermann, Lancet, 1, 1253 (1984); R. Cheingsong-Popov, R. A. Weiss, A. Dalgleish, R. S. Tedder, D. J. Jeffries, D. C. Shannon, R. B. Ferns, E. M. Briggs, I. V. D. Weller, S. Mitton, M. W. Adler, C. Farthing, A. G. Lawrence, B. G. Gazzard, J. Weber, J. R. W. Harris, A. J. Pinching, J. Craske and J. A. J. Barbara, Lancet, 2, 477 (1984); B. Safai, J. E. Groopman, M. Popovic, J. Schupbach, M. G. Sarngadharan, K. Arnett, A. Sliski and R. C. Gallo, Lancet, 1, 1438 (1984)), but such antibodies are not always detectable in infected persons (S. Z. Salahuddin, P. D. Markham, R. R. Redfield, M. Essex, J. E. Groopman, M. G. Sarngadharan, M. F. McLane, A. Sliski and R. C. Gallo, Lancet, 2, 1418 (1984); B. G. Gazzard, C. Farthing, D. C. Shanson, A. G. Lawrence, R. S. Tedder, R. Cheingsong-Popov, A. Dalgleish and R. A. Weiss, Lancet, 2, 480 (1984)).
HTLV III/LAV is rarely transmitted by blood transfusion (J. W. Curran, D. N. Lawrence, H. Jaffe, J. E. Kaplan, L. D. Zyla, M. Chamberland, R. Weinstein, K.-J. Lui, L. B. Schoenberger, T. J. Spira, W. J. Alexander, G. Swinger, A. Ammann, S. Solomon, D. Auerbach, D. Mildvan, R. Stoneburner, J. M. Jason, H. W. Haverkos and B. L. Evatt, N. Engl. J. Med., 310, 69 (1984); H. W. Jaffe, D. P. Francis, M. F. McLane, C. Cabradilla, J. W. Curran, B. W. Kilbourne, D. N. Lawrence, H. W. Haverkos, T. J. Spira, R. Y. Dodd, J. Gold, D. Armstrong, A. Ley, J. Groopman, J. Mullins, T. H. Lee and M. Essex, Science, 223, 1309 (1984); P. M. Feorino, V. S. Kalyanaraman, H. W. Haverkos, C. D. Cabradilla, D. T. Warfield, H. W. Jaffe, A. K. Harrison, M. S. Gottlieb, D. Goldfinger, J.-C. Chermann, F. Barre-Sinoussi, T. T. Spira, J. S. McDougal, J. W. Curran, L. Montagnier, F. A. Murphy and D. P. Francis, Science, 225, 69 (1984); J. E. Groopman, S. Z. Salahuddin, M. G. Sarngadharan, J. I. Mullins, J. L. Sullivan, C. Mulder, C. J. O'Hara, S. H. Cheeseman, H. Haverkos, P. Forgacs, N. Riedel, M. F. McLane, M. Essex and R. C. Gallo, N. Engl. J. Med., 311, 1419 (1984); A. M. Hardy, J. R. Allen, W. M. Morgan and J. W. Curran, JAMA, 253, 215 (1985), and much more frequently by products prepared from pooled human plasma (Vilmer et al., supra; Popovic et al., supra; Cheingsong-Popov et al., supra; B. L. Evatt, D. P. Francis, M. F. McLane, T. H. Lee, C. Cabradilla, S. F. Stein, D. N. Lawrence, J. S. McDougal, T. J. Spira, J. I. Mullens and M. Essex, Lancet, 2, 698 (1983); L. W. Kitchen, F. Barin, J. L. Sullivan, M. F. McLane, D. B. Brettler, P. H. Levine and M. Essex, Nature, 312, 367 (1984); M. Melbye, R. J. Briggar, J. C. Chermann, L. Montagnier, S. Steinbjerg and P. Ebbesen, Lancet, 2, 40 (1984); M. Melbye, R. Madhok, P. S. Sarin, G. D. O. Lowe, J. J. Goedert, K. S. Froebel, R. J. Briggar, S. Stenbjerg, C. D. Forbes, R. C. Gallo and P. Ebbesen, Lancet, 2, 1444 (1984)), as indicated by the high prevalence of anti-HTLV III/LAV antibodies in hemophiliacs. The risk of HTLV III/LAV transmission has indicated the need for screening of blood donors for markers of infection with this human retrovirus(es). Since detection of viral markers (antigens, reverse transcriptase or specific nucleotide sequences) produced by in vitro replicating T lymphocytes, isolated from individual donors, is prohibitively cumbersome for screening purposes, the assay methods have to be based on the presence of antigens or antibodies in serum. Individuals who have been infected with HTLV III or LAV viruses generally may be identified on the basis of a positive serological test for antibodies against the protein components of these viruses. Purified viruses or viral proteins have been heretofore utilized for developing such tests.
The following methods for detection of antibodies to HTLV III/LAV-specific proteins have been developed: (1) an ELISA assay in which wells of polystyrene plates coated with detergent-disrupted purified virus are used, and the presence of antibodies reacting with virus proteins is detected by enzyme-labeled anti-human IgG (C. Saxinger and R. C. Gallo, Lab. Invest., 49, 371 (1983); Sarngadharan et al., supra; Brun-Vezinet et al., supra); (2) Western blot analyses in which viral proteins separated by electrophoresis are transferred to strips of nitrocellulose and reacted with serum specimens; the attached antibodies are subsequently detected with .sup.125 I-labeled anti-human IgG (Schupback et al., supra; Safai et al., supra); (3) a radioimmunoprecipitation test (RIPA), followed by polyacrylamide gel electrophoresis which detects antibodies to [.sup.35 S]-labeled viral proteins (F. Brun-Vezinet, C. Rouzioux, L. Montagnier, S. Chamaret, J. Gruest, F. Barre-Sinoussi, D. Geroldi, J. C. Chermann, J. McCormick, S. Mitchell, P. Piot, H. Taelman, K. B. Mirlangu, M. N. O. W. Mbendi, M. P. K. Kalambayi, C. Bridts, J. Desmyter, F. M. Feinsod and T. C. Quinn, Science, 226, 453 (1984); Kitchen et al., supra); (4) a RIPA test with .sup.125 I-labeled purified LAV core protein P25 (Kalyanaraman et al., supra); (5) an indirect living cell immunofluorescence test of HTLV III-infected cells (Evatt et al., supra; Kitchen et al., supra; Cheingsong-Popov et al., supra); and (6) a competitive RIA test utilizing human anti-HTLV III IgG on a solid support, the same IgG labeled with .sup.125 I, and a crude extract from HTLV III-infected cells (R. S. Tedder, D. C. Shanson, D. J. Jefries, R. Cheingsong-Popov, A. Dalgleish, P. Clapham, K. Nagy and R. A. Weiss, Lancet, 2, 125 (1984); Cheingsong-Popov et al., supra).
Methods (1) and (4) above require either purified virus or a purified isolated viral protein. Virus purification, however, generally involves elaborate, expensive and potentially hazardous virus purification steps. Methods (5) and (6) above are based on the use of infected cells.
For a better understanding of the natural history of HTLV III/LAV infections, it seems important to follow the appearance in serum of antibodies to distinct viral proteins. Methods (2) and (3) above are ideally suited for that purpose. However, these methods are too complex for routine screening purposes. Therefore, simpler direct RIA or ELISA methods for antibodies to distinct epitopes of HTLV III/LAV structural components would be of great value.
Using the double-antibody test (Saxinger and Gallo, supra), proposed for routine screening, 93% of healthy blood donors were negative, 6% appeared borderline positive and 1% appeared positive for anti-HTLV III (S. H. Weiss, J. J. Goedert, M. G. Sarngadharan, A. J. Bodner, R. C. Gallo and W. A. Blattner, JAMA, 253, 221 (1985). This necessitated further confirmatory tests by Western blot analysis, which demonstrated that in most cases of healthy donors, the ELISA-positive samples were actually negative for anti-HTLV III. When other assays were utilized, the prevalence of anti-HTLV III/LAV-positives among healthy donors, not belonging to the high-risk groups of developing AIDS, was 1/298 by indirect membrane immunofluorescence (Jaffe et al., supra), 0/259 by RIPA with .sup.125 I-labeled LAV core protein (Kalyanaraman et al., supra) and 0/1,000 by a competitive RIA utilizing .sup.125 I-labeled anti-HTLV III/LAV (Cheingsong-Popov et al., supra).